JPS5837832B2 - Enzyme adsorbent and enzyme purification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a compound of the general formula (I) (wherein r1, r2 and r3 each represent a hydrogen atom or a methyl group).

The present invention relates to an enzyme adsorbent whose ligand is a compound represented by the following formula, and a method for purifying an enzyme using the adsorbent.

In recent years, various enzyme purification techniques using affinity chromatography have been established, including trypsin, kallikrein,
Regarding the purification of serine proteases such as urokinase, affinity chromatography using various inhibitors as ligands has been reported.

However, the cone is used to obtain a large amount of the inhibitor used as a ligand, the specific affinity for the target enzyme is not sufficient, and the pyrogenic substances mixed in the crude enzyme-containing solution are not completely removed. Many of them have the following drawbacks.

The purpose of the present invention is to develop affinity chromatography that efficiently purifies a large amount of enzyme and has a high pyrogenic substance removal rate.

The present inventors focused on urokinase, which is frequently used as a drug for treating thrombosis and in combination with anticancer drugs, and investigated the search for a ligand that has specific affinity for the enzyme and its affinity chromatography. As a result, it was found that the adsorbent using the compounds represented by the general formula (D) as a ligand not only specifically adsorbs human urokinase, but also has a strong affinity for serine proteases such as trypsin and kallikrein. The present invention was completed based on this discovery.

In the general formula (I), adsorbents having 2,5-monosubstituted, 2,3-mono-substituted, and 1,5-monosubstituted compounds as ligands have particularly excellent adsorption power, and are effective in removing pyrogenic substances. The rate is high;
In addition, the enzyme yield is high.

The compound represented by the above general formula ('I,) is novel *
*It is a compound and is produced according to the reaction formula below.

The reaction was carried out by converting the corresponding nitronaphthoic acid amide into L. Wei
Ntraub et al.'s law (Journal of Organic Chemistry - Vol. 33, p. 679, 1968)
[Step (a)] This nitronaphthamidine is converted into a nitronaphthamidine based on [Step (b)].

Next, the method for producing the compound of the present invention will be explained in further detail with reference to production examples.

Production Example 1 (Synthesis of 5-nitro-1-naphthamidine hydrochloride) 90rfLl of methylene chloride was stirred, 10f (0.046 mol) of 5-nitro-1-naphthoic acid amide was added thereto, and then triethyloquinium fluoroborate was added. 9.
3P (0.0 49 mol) of methylene chloride (25 m
A solution of was added thereto, and the mixture was stirred at room temperature for 15 hours.

The reaction mixture was concentrated to dryness under reduced pressure, and the resulting crystals were washed with dry ether and then dried under reduced pressure.

The total amount of this crystal was dissolved in an ammonia ethanol solution of 17.51 g.
(ammonia content 9%, 0.093 mol),
Stirred at room temperature for 72 hours.

The reaction product was dried under reduced pressure to distill off ammonia and ethanol, 25 TrL of water was added to the residue, stirred, and 48% aqueous sodium hydroxide solution was added to the residue while cooling with water.7. 5f (
0.09 mol) was added thereto, and the resulting crystals were collected and washed with ice-cold water.

The obtained crude 5-nitro-1-naphthamidine was stirred and mixed with 20% hydrochloric acid 12f (0.066 mol), a lot of crystals were collected, and the crystals were stirred and mixed with ethyl acetate 15rrL1.

Washing with ethyl acetate was repeated five times.

The crystals were dried under reduced pressure of phosphorus pentoxide (11 n!ILHg) to constant weight.

Yield of 5-nitro 1-naphthamidine hydrochloride3. Ty
(Yield: 32%) Melting point: 277-279°C, ** single spot on thin layer chromatography.

(b) Synthesis of 5-amino-1-nanotoamidine hydrochloride 5-nitro-1-naphthamidine hydrochloride 3.5f (0.
014 mol) methanol 100rrLl and palladium carbon (palladium content 10%) 0.22 in an internal volume of 30
The mixture was placed in a 0rul pressure glass reactor, and the reactor was purged with nitrogen and then replaced with hydrogen gas, the hydrogen pressure was set to 5k9/c4, and reduction was carried out at room temperature for 5 hours.

The reaction mixture was separated under a nitrogen stream, and the r liquid was concentrated to dryness under reduced pressure.

The obtained crystals were washed with a small amount of cold methanol to obtain 5-amino-1-naphthamidine hydrochloride 2.6P (yield 84%).

Melting point: 230°C (decomposed) Production Example 2 (a) Synthesis of N-methyl-5-nitro-2-naphthamidine hydrochloride 40 m of methylene chloride & stirred, and N-methyl-5-
Nitro 2-naphthoic acid amide 3.6? (0.016 mol) was added thereto, and then a methylene chloride (IQmA) solution of triethyloxonium fluorobole} 3.0 P (0.016 mol) was added, and the mixture was stirred at room temperature for 15 hours.

The reaction mixture was concentrated to dryness under reduced pressure, and the resulting crystals were washed with dry ether and then dried under reduced pressure.

The total amount of this crystal was mixed with 5.8 f of ammonia in ethanol solution (
(Ammonia content: 9%, 0.031 mol) and stirred at room temperature for 72 hours.

The reaction product was dried under reduced pressure, water was added to the residue, stirred, and while cooling on ice, a 5N aqueous sodium hydroxide solution IQ was added.
mA! (0.05 mol) was added, and the formed crystals were collected and then washed with ice-cold water.

The obtained crude N-methyl-5-nitro-2-naphthamidine was stirred and mixed with 20% hydrochloric acid 3S' (0.016 mol), and N-methyl-5-nitro-2-naphthamidine was prepared according to the post-treatment in Example 1. Hydrochloride 2.51 (yield 59%)
I got it.

Melting point 270-272°C (b) Synthesis of N-methyl-5-amino-2-naphthamidine hydrochloride N-methyl-5-nitro-2-naphthamidine hydrochloride 1
．． 40 ml of 7P (6.4 mmol) ethanol and 0.1 f of palladium on carbon (palladium content 10%) were placed in a pressure-resistant glass reactor with an internal volume of 1001717, and reduced according to Example 1 to obtain N-methyl-5-nitro. 1.3 fI (yield 87%) of 2-naphthamidine hydrochloride was obtained.

Melting point 289-292℃ (decomposition) Table 1 shows representative compounds of the present invention.

Intermediates used to prepare the compounds shown in Table 1 are shown in Table 2.

The carrier used in the present invention may be any water-insoluble carrier that can bind the compound represented by the general formula (I) directly or via a spacer, for example,
Agarose, crosslinked dextran, celluloses, polysaccharides such as agar, polymers such as polyacrylamide, and other glass powders can be used, but agarose is usually used.

The adsorbent of the present invention can be obtained by bonding the compound represented by the general formula (I) to the above-mentioned carrier directly or via a spacer in a conventional manner.

For example, when agarose is used as a carrier, the agarose is activated with cyanogen bromide and then reacted with the compound of the general formula (I), or after bonding the group represented by the general formula (I) which is a spacer, The enzyme adsorbent of the present invention can be obtained by reacting the compound of formula (I).

As for the spacer shown in the above general formula, m is usually 3 to 1.
0 is used.

In carrying out the method of the present invention, the adsorbent obtained as described above is packed into a column, and the column is brought into sufficient contact with the adsorbent by a method such as flowing a crude enzyme-containing solution down the column.

After thoroughly washing and removing unadsorbed impurities, the enzyme is eluted using an appropriate eluent.

The crude enzyme-containing solution and eluent to be applied to the column are
Although it is not necessary to limit H to a particularly narrow range, it is adjusted appropriately depending on each enzyme.

In the purification of urokinase, the salt concentration of the crude enzyme-containing solution is 0.2 to 2M, preferably 0.2 to IM, and the pH is 5.
．． 5-10, preferably 6-8.5, the salt concentration of the eluent is 0.5 M or less, and the pH is 3.5-5.5, preferably <<
4-5 acetate buffer is used.

Highly pure enzyme powder can be obtained by treating the eluted enzyme-containing solution with conventional methods such as ultrafiltration and freeze-drying.

Furthermore, as a result of research into advantageous uses of the enzyme adsorbent of the present invention, the present inventors have discovered other chromatography methods that can be used in combination with the chromatography method of the present invention to further enhance purification efficiency. I found a method using graphics.

That is, the crude enzyme-containing solution was prepared using the general formula (■) * (wherein, X represents an amino group or a carboxyl group, and Y represents an alkylamino group, an alkyl-substituted quaternary ammonium, or an alkoxycarbonyl group).

) is passed through a packed bed (1) of a combination of a compound represented by the formula (1) and a water-insoluble carrier, and the effluent from the packed bed (1) is directly transferred to the packed bed (2) of the enzyme adsorbent of the present invention. The enzyme adsorbed on the adsorbent is eluted.

The above-mentioned conjugate of the packed bed (1) is usually formed by bonding a compound represented by the general formula (II) as a ligand to a water-insoluble carrier via a spacer.

For example, the conjugate is one in which a group represented by the general formula (where m is an integer of 3 to 10 and Y is the same as above) is bonded to a water-insoluble injection carrier.

The type of water-insoluble carrier in the conjugate and its manufacturing method are the same as in the case of the enzyme adsorbent of the present invention.

Enzymes to which the purification method of the present invention can be applied include, in addition to urokinase, various serine proteases such as kallikrein, trypsin, chymotrypsin, thrombin, clasmin, and elastase.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 100 ml of Sepharose 4B (trade name, manufactured by Pharmacia Fine Chemicals, Sweden) was activated with cyanogen bromide at pH 11 for 10 minutes, and then the pH
At 9.5, add 5 g of hexamethylene diamine and heat at 4℃ for 20 minutes.
Allowed time to react.

The washed reaction product was suspended in 100 ml of water, succinic anhydride 101 was added, and the pH was adjusted to 6.0 with sodium hydroxide solution.
After reacting for 7 hours while maintaining the temperature, the mixture was washed with 1 liter of water.

Add 77 liters of distilled water to 10 TIL of the product to suspend it, add 60 μl of 5-amino-2-naphthamidine hydrochloride to this under stirring, and keep the pH of the liquid at 4.7 with 1N hydrochloric acid or 1N sodium hydroxide. , and further l-ethyl-3-
A solution prepared by dissolving 570 μl of (3-dimethylaminopropyl)carbodiimide hydrochloride in 1 part water was added over 5 minutes, and stirring was continued at room temperature of 20° C. for 20 hours.

After filtering the reaction product, 0.01N hydrochloric acid, 0.0 IN
Sodium hydroxide, IM sodium monochloride aqueous solution, 0.
1M = acetate buffer, IM sodium monochloride aqueous solution, 0.
1 M Tris buffer (pH 8.0), followed by **
The enzyme adsorbent of the present invention was obtained by washing with IM sodium monochloride aqueous solution once and bonding to agarose with OOml.

Example 2 5-amino-1-naphthamidine hydrochloride 60m instead of the 5-amino-2-naphthamidine salt** acid salt in Example 1
9 was added and treated in the same manner as in Example 1 to obtain the enzyme adsorbent of the present invention which is bound to agarose.

Example 3 1 ml of the adsorbent obtained in Example 1 was packed into an affinity column packed in a plastic tube with a diameter of 5 mm, and 4 ml of a crude urokinase solution obtained from human urine was added using Celite as an adsorbent.
urokinase (containing 2000 international units, specific activity 5000 international units/m9 protein), then 8 ml of 0.3 M
- 0.1M phosphate buffer containing NaCl (pH 7.
5), 121IL! ! 0 containing 0.4M NaCl
．． 1M acetate buffer (pH 4.0), 12 rul.
0.1 M acetate buffer (p
H4.0), and further 4 ml of 6M urea aqueous solution (pH4.0).
) were sequentially flowed down.

The absorbance at 280 nm and urokinase activity of the effluent are shown in FIG.

From FIG. 1, it was found that the urokinase activity per protein unit was purified approximately 10 times.

Example 4 Using the adsorbent obtained in Example 2, crude urokinase was treated and purified in the same manner as in Example 3.

Uyukinase activity per protein unit was purified approximately 10 times.

Example 5 Conjugate 1 obtained by bonding a group represented by the formula to Sepharose 4B (trade name, manufactured by Pharmacia Fine Chemicals) by a conventional method
0 ml was packed into a column with a diameter of 0.92 cfrL (hereinafter referred to as A column).

Separately, the adsorbent 10 mJ obtained in Example 1 was prepared with a diameter of 0.92 mm.
It was packed into a crIL column (hereinafter referred to as B column).

Crude urokinase obtained from human urine using Celite as an adsorbent (
Contains urokinase 1, O x 106 international units, specific activity 5000 international units/1r&? protein), 0.0.0% containing 0.4M sodium chloride. 1 M phosphate buffer (pH 7.
0) in 50 ml and passed through the A column, then 0.0 ml containing 0.4 M sodium chloride. 1M IJ
Acid buffer (pH 7.0) was passed through the A column.

During this time, the effluent from the A column was directly passed through the B column.

Next, after washing the B column with IM sodium chloride-containing phosphate buffer (pH 7.5), urokinase was eluted by flowing 0.1M acetate buffer (pH 4, 5) containing 1.5M sodium chloride. Ta.

Urokinase active fractions were collected, dialysis concentrated, lyophilized,
Urokinase powder (specific activity: 113,500 international units/■ protein) with a concentration of 0.75 x lO' international units was obtained.

The urokinase obtained in Examples 3, 4, and 5 was negative in the pyrogen test at a dose of 8000 international units/kg of rabbit body weight.

[Brief explanation of the drawing]

Figure 1 shows the absorbance (280 nm) of the effluent in Example 3.
) and urokinase activity.

Claims (1)

[Scope of Claims] 1. An enzyme adsorbent whose sagand is a compound represented by the general formula (wherein r1, r2 and r3 each represent a hydrogen atom or a methyl group).